It has been required in various fields such as clinical treatment, food, an environment, and the like to detect a target. Interactions with a target are generally utilized to detect the target. Among them, a method using an antibody that specifically binds to a target has been widely used. In this method, the target is bound to an antibody labeled with oxidoreductase such as peroxidase, for example. Then, a chromogenic reaction is performed by an enzyme in the labeled antibody using a chromogenic substrate. The target is indirectly subjected to analysis such as, for example, qualitative analysis or quantitative analysis by the detection of color development.
However, the antibody is obtained by immunization of animals. Thus, it is difficult to obtain a specific antigen to a highly toxic target or a low-molecular-weight target. Therefore, a nucleic acid molecule that binds to a target, i.e., a nucleic acid aptamer (hereinafter also referred to as an “aptamer”) has received attention in recent years. The aptamer can be obtained in a test tube. Thus, for example, the aptamers to a toxic target and a low-molecular-weight target can be obtained.
In order to use such aptamer in detection of a target as a substitute for the antibody, it is attempted to use the aptamer in combination with DNAzyme that exhibits the same catalytic activity as in peroxidase. The DNAzyme generally has a guanine-rich structural motif and has a G-quadruplex structure and is DNA that exhibits a catalyst function of peroxidase by forming a complex through binding with hemin.
In the detection of a target, a single-stranded nucleic acid element obtained by linking a single-stranded aptamer and a single-stranded DNAzyme is specifically utilized (Non-Patent Document 1). The single-stranded nucleic acid element forms a stem structure by self-annealing in the absence of a target, and by the stem structure, the DNAzyme has a structure of not being capable of forming in G-quadruplex. Thus, in the absence of a target, DNAzyme in the single-stranded nucleic acid element cannot bind to hemin and thus cannot exhibit a catalyst function. On the other hand, in the presence of a target, the single-stranded nucleic acid element releases its stem structure by binding of the target to the aptamer. Thus, in the presence of a target, the DNAzyme in the single-stranded nucleic acid element forms G-quadruplex and exhibits a catalyst function by binding of the target to hemin. Therefore, when a chromogenic substrate to the redox activity is present together, a chromogenic reaction is generated in the presence of a target and is not generated in the absence of a target. Thus, the target can be analyzed by detecting the chromogenic reaction. Furthermore, it is not required to label the target, and thus, a wide range of targets including a low-molecular-weight substance and the like can be subjected to direct detection.
Therefore, a method in which DNAzyme superior in catalyst function and an aptamer showing superior binding force or superior specificity to the target are selected, and both are linked to produce a single-stranded nucleic acid element is employed. However, even when the selected DNAzyme and the selected aptamer are linked, it is really difficult to obtain a nucleic acid element suitable for practical use, for example, the nucleic acid element having a sufficient ratio (S/N) ratio between activity (S) in the presence of the target and activity (N) in the absence of the target. Therefore, it is unknown whether or not the element having sufficient S/N ratio is obtained even by modifying both of the DNAzyme and the aptamer to various sequences and producing a large number of nucleic acid element candidates from these combinations.
For these reasons, it is required to establish a method for simply and efficiently screening nucleic acid element candidate molecules for a single-stranded nucleic acid element superior in S/N ratio.